Branching mode in complex-type triantennary N-glycans as regulatory element of their ligand properties

We address the question whether the two natural types of branching in complex-type triantennary N-glycans differ in ligand properties. Toward this end, we prepared the set of pergalactosylated undecasaccharides and derivatives with alpha2,3/6-sialylation by chemoenzymatic synthesis. Conjugation resulted in neoglycoproteins which were tested in assays with lectins/antibodies, cultured cells and animals. Solid-phase assays with galactoside-specific proteins (a plant toxin, galectins and an antibody fraction) disclosed that the branching mode did not significantly affect affinity. However, compared to previous studies under identical conditions increase in antennae number and presence of substitutions in biantennary N-glycans altered KD-values with differences between receptors. Neoglycoprotein binding to cells of eight human tumor lines was sensitive to N-glycan branching. Staining intensity revealed pronounced branch-mode-dependent differences in four cases. Biodistribution profiles in mice uncovered dramatic changes in clearance rates with prolonged serum presence associated with type II branching of sialylated N-glycans and markedly increased uptake of neoglycoproteins with type I-branched N-glycans into liver, spleen, heart and lungs. This part of the study is relevant for rational glycoengineering of pharmaproteins. In general, our study supports the concept to view details of N-glycan structure, here branching, as a means to modulate ligand properties.     

Substitutions in the N-glycan core as regulators of biorecognition: the case of core-fucose and bisecting GlcNAc moieties

Core fucosylation and the bisecting N-acetylglucosamine residue are prominent natural substitutions of the N-glycan core. To address the issue of whether these two substituents can modulate ligand properties of complex-type biantennary N-glycans, we performed chemoenzymatic synthesis of the respective galactosylated and alpha2,3/6-sialylated N-glycans. Neoglycoproteins were then produced to determine these glycans' reactivities with sugar receptors in solid-phase assays and with tumor cells in vitro as well as their in vivo biodistribution profiles in mice. Slight protein-type-dependent changes were noted in lectin binding, including adhesion/growth-regulatory galectins as study objects, when the data were related to properties of N-glycans without or with only one core substituent. Monitoring binding in vitro revealed cell-type-dependent changes. They delimited the ligand activity of this glycan type from that of chains with un- and monosubstituted cores. A markedly prolonged serum half-life was conferred to the neoglycoprotein by the galactose-terminated N-glycan, which together with increased organ retention of all three neoglycoproteins underscores the conspicuous relevance for glycoengineering of pharmaproteins. The predominant presentation of the two branches in the disubstituted N-glycan as extended (alpha1,3-antenna) and backfolded (alpha1,6-antenna) forms, revealed by molecular dynamics simulations, can underlie the measured characteristics. These results obtained by a combined strategy further support the concept of viewing N-glycan core substitutions as non-random additions which exert a modulatory role on ligand properties. Moreover, our data inspire us to devise new, non-natural modifications to realize the full potential of glycoengineering for diagnostic and therapeutic purposes.     

Carbohydrate specificity of lectins from Boletopsis leucomelas and Aralia cordate

The carbohydrate specificity of three novel lectins, Boletopsis leucomelas lectin (BLL), Aralia cordate lectin (ACL), and Wasabia japonica lectin (WJL), was examined by frontal affinity chromatography using a panel of fluorescently labeled 47 oligosaccharides. The results indicate that BLL recognizes an agalacto structure of the biantennary chain and its bisecting structure. ACL showed strong affinity for triantennary oligosaccharides, but no affinity for tetraantennary structure. WJL showed no appreciable affinity for any of the 47 glycans examined. These lectins with a unique affinity specificity might be useful for examining alterations in the glycan structures of the glycoconjugates in association with development and various diseases.     

Carbohydrate Specificity of Lectins from Boletopsis leucomelas and Aralia cordate

The carbohydrate specificity of three novel lectins, Boletopsis leucomelas lectin (BLL), Aralia cordate lectin (ACL), and Wasabia japonica lectin (WJL), was examined by frontal affinity chromatography using a panel of fluorescently labeled 47 oligosaccharides. The results indicate that BLL recognizes an agalacto structure of the biantennary chain and its bisecting structure. ACL showed strong affinity for triantennary oligosaccharides, but no affinity for tetraantennary structure. WJL showed no appreciable affinity for any of the 47 glycans examined. These lectins with a unique affinity specificity might be useful for examining alterations in the glycan structures of the glycoconjugates in association with development and various diseases.     

Specificity of DC-SIGN for mannose- and fucose-containing glycans

The dendritic cell specific C-type lectin dendritic cell specific ICAM-3 grabbing non-integrin (DC-SIGN) binds to "self" glycan ligands found on human cells and to "foreign" glycans of bacterial or parasitic pathogens. Here, we investigated the binding properties of DC-SIGN to a large array of potential ligands in a glycan array format. Our data indicate that DC-SIGN binds with K(d)<2muM to a neoglycoconjugate in which Galbeta1-4(Fucalpha1-3)GlcNAc (Le(x)) trisaccharides are expressed multivalently. A lower selective binding was observed to oligomannose-type N-glycans, diantennary N-glycans expressing Le(x) and GalNAcbeta1-4(Fucalpha1-3)GlcNAc (LacdiNAc-fucose), whereas no binding was observed to N-glycans expressing core-fucose linked either alpha1-6 or alpha1-3 to the Asn-linked GlcNAc of N-glycans. These results demonstrate that DC-SIGN is selective in its recognition of specific types of fucosylated glycans and subsets of oligomannose- and complex-type N-glycans.     

Control of glycoprotein synthesis. Bovine milk UDPgalactose:N-acetylglucosamine beta-4-galactosyltransferase catalyzes the preferential transfer of galactose to the GlcNAc beta 1,2Man alpha 1,3- branch of both bisected and nonbisected complex biantennary asparagine-linked oligosaccharides

Bovine milk UDPgalactose:N-acetylglucosamine beta-4-galactosyltransferase has been used to investigate the effect of a bisecting GlcNAc residue (linked beta 1,4 to the beta-linked mannose of the trimannosyl core of asparagine-linked complex oligosaccharides) on galactosylation of biantennary complex oligosaccharides. Columns of immobilized lectins (concanavalin A, erythroagglutinating phytohemagglutinin, and Ricinus communis agglutinin 120) were used to separate the various products of the reactions. Preferential galactosylation of the GlcNAc beta 1,2Man alpha 1,3 arm occurred both in the absence and in the presence of a bisecting GlcNAc residue; the ratio of the rates of galactosylation of the Man alpha 1,3 arm to the Man alpha 1,6 arm was 6.5 in the absence of a bisecting GlcNAc and 2.8 in its presence. The bisecting GlcNAc residue reduced galactosylation of the Man alpha 1,3 arm by about 78% probably due to steric hindrance of the GlcNAc beta 1,2Man alpha 1,3 beta 1,4 region of the substrate by the bisecting GlcNAc. This steric hindrance prevents the action of four other enzymes involved in assembly of complex asparagine-linked oligosaccharides and indicates the importance of the bisecting GlcNAc residue in the control of glycoprotein biosynthesis. The Man alpha 1,3 arm of biantennary oligosaccharides is believed to be freely accessible to enzyme action whereas the Man alpha 1,6 arm is believed to be folded back toward the core. This may explain the preferential action of Gal-transferase on the Man alpha 1,3 arm of both bisected and nonbisected oligosaccharides.     

Analysis of the site-specific N-glycosylation of beta1,6 N-acetylglucosaminyltransferase V

N-acetylglucosaminyltransferase V (GnT-V) catalyzes the addition of a beta1,6-linked GlcNAc to the alpha1,6 mannose of the trimannosyl core to form tri- and tetraantennary N-glycans and contains six putative N-linked sites. We used mass spectrometry techniques combined with exoglycosidase digestions of recombinant human GnT-V expressed in CHO cells, to identify its N-glycan structures and their sites of expression. Release of N-glycans by PNGase F treatment, followed by analysis of the permethylated glycans using MALDI-TOF MS, indicated a range of complex glycans from bi- to tetraantennary species. Mapping of the glycosylation sites was performed by enriching for trypsin-digested glycopeptides, followed by analysis of each fraction with Q-TOF MS. Predicted tryptic glycopeptides were identified by comparisons of theoretical masses of peptides with various glycan masses to the masses of the glycopeptides determined experimentally. Of the three putative glycosylation sites in the catalytic region, peptides containing sites Asn 334, 433, and 447 were identified as being N-glycosylated. Asn 334 is glycosylated with only a biantennary structure with one or two terminating sialic acids. Sites Asn 433 and 447 both contain structures that range from biantennary with two sialic acids to tetraantennary terminating with four sialic acids. The predominant glycan species found on both of these sites is a triantennary with three sialic acids. The appearance of only biantennary glycans at site Asn 433, coupled with the appearance of more highly branched structures at Asn 334 and 447, demonstrates that biantennary acceptors present at different sites on the same protein during biosynthesis can differ in their accessibility for branching by GnT-V.     

N-glycan structures of human transferrin produced by Lymantria dispar (gypsy moth) cells using the LdMNPV expression system

N-glycan structures of recombinant human serum transferrin (hTf) expressed by Lymantria dispar (gypsy moth) 652Y cells were determined. The gene encoding hTf was incorporated into a Lymantria dispar nucleopolyhedrovirus (LdMNPV) under the control of the polyhedrin promoter. This virus was then used to infect Ld652Y cells, and the recombinant protein was harvested at 120 h postinfection. N-glycans were released from the purified recombinant human serum transferrin and derivatized with 2-aminopyridine; the glycan structures were analyzed by a two-dimensional HPLC and MALDI-TOF MS. Structures of 11 glycans (88.8% of total N-glycans) were elucidated. The glycan analysis revealed that the most abundant glycans were Man1-3(+/-Fucalpha6)GlcNAc2 (75.5%) and GlcNAcMan3(+/-Fucalpha6)GlcNAc2 (7.4%). There was only approximately 6% of high-mannose type glycans identified. Nearly half (49.8%) of the total N-glycans contained alpha(1,6)-fucosylation on the Asn-linked GlcNAc residue. However alpha(1,3)-fucosylation on the same GlcNAc, often found in N-glycans produced by other insects and insect cells, was not detected. Inclusion of fetal bovine serum in culture media had little effect on the N-glycan structures of the recombinant human serum transferrin obtained.     

Immunoglobulin G Fc N-glycan profiling in patients with gastric cancer by LC-ESI-MS: relation to tumor progression and survival

The IgG Fc glycans strongly influence the Fcγ receptor interactions and Fc-mediated effector mechanisms. Changes in the structure of IgG glycans are associated with various diseases, such as infections and autoimmunity. However, the possible role of Fc glycans in tumor immunity is not yet fully understood. The aim of this study was to profile the Fc N-glycans of IgG samples from patients with gastric cancer (n = 80) and controls (n = 51) using LC-ESI-MS method to correlate the findings with stage of cancer and patients survival. Analysis of 32 different IgG N-glycans revealed significant increase of agalactosylated (GnGnF, GnGn(bi)F), and decrease of galactosylated (AGn(bi), AGn(bi)F, AA(bi), AAF) and monosialylated IgG glycoforms (NaAF, NaA(bi)) in cancer patients. A statistically significant increase of Fc fucosylation was observed in tumor stage II and III whereas reverse changes were found for the presence of bisecting GlcNAc. Higher level of fully sialylated glycans and elevated expression of glycans with bisecting GlcNAc were associated with better survival rate. Our findings provide the first evidence that the changes in Fc glycan profile may predict the survival of patients with gastric cancer. Cancer stage-dependent changes in Fc fucosylation and the bisecting N-acteylglucosamine expression as well as an association of several IgG glycoforms with the survival suggest that IgG glycosylation is related to pathogenesis of cancer and progression of the disease.     

Introduction of extended LEC14-type branching into core-fucosylated biantennary N-glycan

A series of enzymatic substitutions modifies the basic structure of complex-type biantennary N-glycans. Among them, a beta1,2-linked N-acetylglucosamine residue is introduced to the central mannose moiety of the core-fucosylated oligosaccharide by N-acetylglucosaminyltransferase VII. This so-called LEC14 epitope can undergo galactosylation at the beta1,2-linked N-acetylglucosamine residue. Guided by the hypothesis that structural modifications in the N-glycan alter its capacity to serve as ligand for lectins, we prepared a neoglycoprotein with the extended LEC14 N-glycan and tested its properties in three different assays. In order to allow comparison to previous results on other types of biantennary N-glycans the functionalization of the glycans for coupling and assay conditions were deliberately kept constant. Compared to the core-fucosylated N-glycan no significant change in affinity was seen when testing three galactoside-specific proteins. However, cell positivity in flow cytofluorimetry was enhanced in six of eight human tumor lines. Analysis of biodistribution in tumor-bearing mice revealed an increase of blood clearance by about 40%, yielding a favorable tumor/blood ratio. Thus, the extended LEC14 motif affects binding properties to cellular lectins on cell surfaces and organs when compared to the core-fucosylated biantennary N-glycan. The results argue in favor of the concept of viewing substitutions as molecular switches for lectin-binding affinity. Moreover, they have potential relevance for glycoengineering of reagents in tumor imaging.     

The bisecting GlcNAc in cell growth control and tumor progression

The bisecting GlcNAc is transferred to the core mannose residue of complex or hybrid N-glycans on glycoproteins by the β1,4-N-acetylglucosaminyltransferase III (GlcNAcT-III) or MGAT3. The addition of the bisecting GlcNAc confers unique lectin recognition properties to N-glycans. Thus, LEC10 gain-of-function Chinese hamster ovary (CHO) cells selected for the acquisition of ricin resistance, carry N-glycans with a bisecting GlcNAc, which enhances the binding of the erythroagglutinin E-PHA, but reduces the binding of ricin and galectins-1, -3 and -8. The altered interaction with galactose-binding lectins suggests that the bisecting GlcNAc affects N-glycan conformation. LEC10 mutants expressing polyoma middle T antigen (PyMT) exhibit reduced growth factor signaling. Furthermore, PyMT-induced mammary tumors lacking MGAT3, progress more rapidly than tumors with the bisecting GlcNAc on N-glycans of cell surface glycoproteins. In recent years, evidence for a new paradigm of cell growth control has emerged involving regulation of cell surface residency of growth factor and cytokine receptors via interactions and cross-linking of their branched N-glycans with a lattice of galectin(s). Specific cross-linking of glycoprotein receptors in the lattice regulates their endocytosis, leading to effects on growth factor-induced signaling. This review will describe evidence that the bisecting GlcNAc of N-glycans regulates cellular signaling and tumor progression, apparently through modulating N-glycan/galectin interactions.     

Recognition of Bisecting N-Acetylglucosamine: STRUCTURAL BASIS FOR ASYMMETRIC INTERACTION WITH THE MOUSE LECTIN DENDRITIC CELL INHIBITORY RECEPTOR 2*

Dendritic cell inhibitory receptor 2 (DCIR2) is a C-type lectin expressed on classical dendritic cells. We recently identified the unique ligand specificity of mouse DCIR2 (mDCIR2) toward biantennary complex-type glycans containing bisecting N-acetylglucosamine (GlcNAc). Here, we report the crystal structures of the mDCIR2 carbohydrate recognition domain in unliganded form as well as in complex with an agalactosylated complex-type N-glycan unit carrying a bisecting GlcNAc residue. Bisecting GlcNAc and the α1-3 branch of the biantennary oligosaccharide asymmetrically interact with canonical and non-canonical mDCIR2 residues. Ligand-protein interactions occur directly through mDCIR2-characteristic amino acid residues as well as via a calcium ion and water molecule. Our structural and biochemical data elucidate for the first time the unique binding mode of mDCIR2 for bisecting GlcNAc-containing glycans, a mode that contrasts sharply with that of other immune C-type lectin receptors such as DC-SIGN.     

Glycan analysis of the chicken synaptic plasma membrane glycoproteins--a major synaptic N-glycan carries the LewisX determinant

The majority of synaptic plasma membrane components are glycosylated. It is now widely accepted that this post-translational modification is crucial during the establishment, maintenance and function of the nervous system. Despite its significance, structural information about the glycosylation of nervous system specific glycoproteins is very limited. In the present study the major glycan structures of the chicken synaptic plasma membrane (SPM) associated glycoprotein glycans were determined. N-glycans were released by hydrazinolysis, labelled with 2-aminobenzamide, treated with neuraminidase and subsequently fractionated by size exclusion chromatography. Individual fractions were characterized by the combination of high-pressure liquid chromatography, exoglycosidase treatment or reagent array analysis method (RAAM). In addition to oligomannose-type glycans, core-fucosylated complex glycans with biantennary bisecting glycans carrying the LewisX epitope were most abundant. The overall chicken glycan profile was strikingly similar to the rat brain glycan profile. The presence of the LewisX determinant in relatively large proportions suggests a tissue-specific function for these glycans.     

Psathyrella velutina Mushroom Lectin Exhibits High Affinity toward Sialoglycoproteins Possessing Terminal N-Acetylneuraminic Acid alpha 2,3-Linked to Penultimate Galactose Residues of Trisialyl N-Glycans. Comparison with other sialic acid-specific lectins

A lectin from the fruiting body of the Psathyrella velutina mushroom (PVL) was found to bind specifically to N-acetylneuraminic acid, as well as to GlcNAc (Ueda, H., Kojima, K., Saitoh, T., and Ogawa, H. (1999) FEBS Lett. 448, 75-80). In this study, the glycan sequences that PVL recognizes with high affinity on sialoglycoproteins were revealed. Among sialic acid-specific lectins only PVL could reveal the sialylated N-acetyllactosamine structure of glycoproteins in blotting studies, based on the dual specificity. The affinity of PVL to fetuin was measured by surface plasmon resonance to be 10(7) m(-1), which is an order of magnitude higher than those of Sambucus nigra agglutinin and Maackia amurensis mitogen, whereas affinity to asialofetuin was approximately 0 and to asialo-agalactofetuin was 10(8) m(-1), suggesting that PVL exhibits remarkably high affinities toward glycoproteins possessing trisialo- or GlcNAc-exposed glycans. Transferrin was separated into fractions that correspond to the sialylation states on an immobilized PVL column. Transferrin-possessing trisialoglycans containing alpha2,3-linked N-acetylneuraminic acid on the beta1,4-linked GlcNAc branch bound to the PVL column and eluted with GlcNAc; those containing only alpha2,6-linked sialic acids were retarded, whereas other transferrin fractions passed through the column. These results indicate that PVL is a lectin with potential for separation and detection of sialoglycoproteins because of its dual specificity toward sialoglycans and GlcNAc exposed glycans.     

N-glycan structures from the major glycoproteins of pigeon egg white: predominance of terminal Galalpha(1)Gal

N-Glycans from major glycoproteins of pigeon egg white (ovotransferrin, ovomucoid, and ovalbumins) were enzymatically released and were reductively aminated with 2-aminopyridine, separated, and structurally characterized by mass spectrometry and a three-dimensional mapping technique using three different columns of high performance liquid chromatography (HPLC) (Takahashi, N., Nakagawa, H., Fujikawa, K., Kawamura, Y., and Tomiya, N. (1995) Anal. Biochem. 226, 139-146). Twenty-five major N-glycan structures, all of them hitherto unknown, were identified as pyridylamino derivatives. Of these, 13 were neutral, 10 were monosialyl, and 2 were disialyl oligosaccharides. All N-glycans contain from one to four Galalpha(1,4)Galbeta(1,4) sequences at the nonreducing terminal positions and are devoid of fucose residues. N-Acetylneuraminic acids were alpha(2,6)-linked only to beta-galactose. The HPLC profiles of the N-glycans from four different glycoproteins were qualitatively very similar to each other, but not identical in the peak distributions. Monosialyl glycans were most abundant in all four glycoproteins, followed by neutral glycans. Disialyl glycans were lowest in ovotransferrin, and highest in ovomucoid. Triantennary structures with bisecting GlcNAc were predominant in ovotransferrin, and tetra-antennary (with and without bisecting GlcNAc-containing) structures were predominant in other glycoproteins. Penta-antennary structures (with a sialic acid and without bisecting GlcNAc residue) were also found in small quantities in all four glycoproteins. In contrast to the chicken egg white counterparts, which contain mostly high mannose and hybrid types, all N-glycan structures in the major pigeon egg white glycoproteins are complex type.     

Structural diversity and changes in conformational equilibria of biantennary complex-type N-glycans in water revealed by replica-exchange molecular dynamics simulation

Structural diversity of N-glycans is essential for specific binding to their receptor proteins. To gain insights into structural and dynamic aspects in atomic detail not normally accessible by experiment, we here perform extensive molecular-dynamics simulations of N-glycans in solution using the replica-exchange method. The simulations show that five distinct conformers exist in solution for the N-glycans with and without bisecting GlcNAc. Importantly, the population sizes of three of the conformers are drastically reduced upon the introduction of bisecting GlcNAc. This is caused by a local hydrogen-bond rearrangement proximal to the bisecting GlcNAc. These simulations show that an N-glycan modification like the bisecting GlcNAc selects a certain “key” (or group of “keys”) within the framework of the “bunch of keys” mechanism. Hence, the range of specific glycan-protein interactions and affinity changes need to be understood in terms of the structural diversity of glycans and the alteration of conformational equilibria by core modification.     

High throughput isolation and glycosylation analysis of IgG-variability and heritability of the IgG glycome in three isolated human populations

All immunoglobulin G molecules carry N-glycans, which modulate their biological activity. Changes in N-glycosylation of IgG associate with various diseases and affect the activity of therapeutic antibodies and intravenous immunoglobulins. We have developed a novel 96-well protein G monolithic plate and used it to rapidly isolate IgG from plasma of 2298 individuals from three isolated human populations. N-glycans were released by PNGase F, labeled with 2-aminobenzamide and analyzed by hydrophilic interaction chromatography with fluorescence detection. The majority of the structural features of the IgG glycome were consistent with previous studies, but sialylation was somewhat higher than reported previously. Sialylation was particularly prominent in core fucosylated glycans containing two galactose residues and bisecting GlcNAc where median sialylation level was nearly 80%. Very high variability between individuals was observed, approximately three times higher than in the total plasma glycome. For example, neutral IgG glycans without core fucose varied between 1.3 and 19%, a difference that significantly affects the effector functions of natural antibodies, predisposing or protecting individuals from particular diseases. Heritability of IgG glycans was generally between 30 and 50%. The individual's age was associated with a significant decrease in galactose and increase of bisecting GlcNAc, whereas other functional elements of IgG glycosylation did not change much with age. Gender was not an important predictor for any IgG glycan. An important observation is that competition between glycosyltransferases, which occurs in vitro, did not appear to be relevant in vivo, indicating that the final glycan structures are not a simple result of competing enzymatic activities, but a carefully regulated outcome designed to meet the prevailing physiological needs.     

Comparative analysis by frontal affinity chromatography of oligosaccharide specificity of GlcNAc-binding lectins, Griffonia simplicifolia lectin-II (GSL-II) and Boletopsis leucomelas lectin (BLL)

Lectin-based structural glycomics requires a search for useful lectins and their biochemical characterization to profile complex features of glycans. In this paper, two GlcNAc-binding lectins are reported with their detailed oligosaccharide specificity. One is a classic plant lectin, Griffonia simplicifolia lectin-II (GSL-II), and the other is a novel fungal lectin, Boletopsis leucomelas lectin (BLL). Their sugar-binding specificity was analyzed by frontal affinity chromatography using 146 glycans (125 pyridylaminated and 21 p-nitrophenyl saccharides). As a result, it was found that both GSL-II and BLL showed significant affinity toward complex-type N-glycans, which are either partially or completely agalactosylated. However, their branch-specific features differed significantly: GSL-II strongly bound to agalacto-type, tri- or tetra-antennary N-glycans with its primary recognition of a GlcNAc residue transferred by GlcNAc-transferase IV, while BLL preferred N-glycans with fewer branches. In fact, the presence of a GlcNAc residue transferred by GlcNAc-transferase V abolishes the binding of BLL. Thus, GSL-II and BLL forms a pair of complementally probes to profile a series of agalacto-type N-glycans.     

Biological consequences of overexpressing or eliminating N-acetylglucosaminyltransferase-TIII in the mouse

N-acetylglucosaminyltransferase III (GlcNAc-TIII), a product of the human MGAT3 gene, was discovered as a glycosyltransferase activity in hen oviduct. GlcNAc-TIII transfers GlcNAc in beta4-linkage to the core Man of complex or hybrid N-glycans, and thereby alters not only the composition, but also the conformation of the N-glycan. The dramatic consequences of the addition of this bisecting GlcNAc residue are reflected in the altered binding of lectins that recognize Gal residues on N-glycans. Changes in GlcNAc-TIII expression correlate with hepatoma and leukemia in rodents and humans, and the bisecting GlcNAc on Asn 297 of human IgG antibodies enhances their effector functions. Overexpression of a cDNA encoding GlcNAc-TIII alters growth control and cell-cell interactions in cultured cells, and in transgenic mice. While mice lacking GlcNAc-TIII are viable and fertile, they exhibit retarded progression of diethylnitrosamine (DEN)-induced liver tumors. Further biological functions of GlcNAc-TIII are expected to be uncovered as mice with a null mutation in the Mgat3 gene are challenged.     

Histochemical analysis of glycoconjugates in the domestic cat testis

The localization and characterization of oligosaccharide sequences in the cat testis was investigated using 12 lectins in combination with the beta-elimination reaction, N-Glycosidase F and sialidase digestion. Leydig cells expressed O-linked glycans with terminal alphaGalNAc (HPA reactivity) and N-glycans with terminal/internal alphaMan (Con A affinity). The basement membrane showed terminal Neu5Acalpha2,6Gal/GalNAc, Galbeta1,3GalNAc, alpha/betaGalNAc, and GlcNAc (SNA, PNA, HPA, SBA, GSA II reactivity) in O-linked oligosaccharides, terminal Galbeta1,4GlcNAc (RCA120 staining) and alphaMan in N-linked oligosaccharides; in addition, terminal Neu5acalpha2,3Galbeta1,4GlcNac, Forssman pentasaccharide, alphaGal, alphaL-Fuc and internal GlcNAc (MAL II, DBA, GSA I-B4, UEA I, KOH-sialidase-WGA affinity) formed both O- and N-linked oligosaccharides. The Sertoli cells cytoplasm contained terminal Neu5Ac-Galbeta1,4GlcNAc, Neu5Ac-betaGalNAc as well as internal GlcNAc in O-linked glycans, alphaMan in N-linked glycoproteins and terminal Neu5Acalpha2,6Gal/ GalNAc in both O- and N-linked oligosaccharides. Spermatogonia exhibited cytoplasmic N-linked glycoproteins with alphaMan residues. The spermatocytes cytoplasm expressed terminal Neu5Acalpha2,3Galbeta1,4 GlcNAc and Galbeta1,3GalNAc in O-linked oligosaccharides, terminal Galbeta1,4GlcNAc and alpha/betaGalNAc in N-linked glycoconjugates. The Golgi region showed terminal Neu5Acalpha2,3Galbeta1,4GlcNac, Galbeta1,4GlcNAc, Forssman pentasaccharide, and alphaGalNAc in O-linked oligosaccharides, alphaMan and terminal betaGal in N-linked oligosaccharides. The acrosomes of Golgi-phase spermatids expressed terminal Galbeta1,3GalNAc, Galbeta1,4GlcNAc, Forssmann pentasaccharide, alpha/betaGalNAc, alphaGal and internal GlcNAc in O-linked oligosaccharides, terminal alpha/betaGalNAc, alphaGal and terminal/internal alphaMan in N-linked glycoproteins. The acrosomes of cap-phase spermatids lacked internal Forssman pentasaccharide and alphaGal, while having increased alpha/betaGalNAc. The acrosomes of elongated spermatids did not show terminal Galbeta1,3GalNAc, displayed terminal Galbeta1,4GlcNAc and alpha/betaGalNAc in N-glycans and Neu5Ac-Galbeta1,3GalNAc in O-linked oligosaccharides.